Vibrational (e.g. Raman) spectroscopy has been used to identify micro-organisms at various taxonomic levels including species level, subspecies level and strain level. Subspecies level typing of micro-organisms enables epidemiological studies. For example, it can be used to determine that different patients in a hospital have been infected with the same micro-organism, which may point at a common source of infection (e.g. a contaminated instrument). This will then enable targeted hygienic measures to be taken. It could also point out that a microbial strain has been transmitted between patients and that measures such as isolation of infected patients may be required. It is estimated that in the industrialized world about 5 to 10% of the patients admitted to a hospital will develop a microbial infection during their stay. These nosocomial or hospital acquired infections are the cause of much patient suffering and place a high financial burden on the health care system. It has been shown that programs based on routine subspecies typing of patient material, by means of which contaminations or outbreaks of infections can be recognized at an early stage, can be very cost-effective. However, such programs are not in place in most hospitals and routine identification of micro-organisms is essentially limited to the species level, which does not provide the epidemiological information to enable early detection of outbreaks and contaminations.
Current methods for microbial typing, both genotyping and phenotyping methods, are not suitable for routine application in hospitals. They are generally costly, have a sample throughput that is too low to keep up with the flow of patient samples in a hospital, and require expert operators and special facilities.
Vibrational spectroscopy does not have these drawbacks and is a good candidate for routine microbial typing. However identification at strain level puts high demands on the reproducibility of the Raman measurements, due to the fact that the spectral differences between closely related strains can be very small. For example, it has been found that there can be greater than 99.9% correlation between Raman spectra of closely related strains. This implies that technology protocols for sample preparation and measurement, and data analysis algorithms used to obtain and analyze Raman spectra of microbial strains must result in a reproducibility of better than 99.9%.
Escoriza et al. (Appl. Spectrosc. 2006, 60(9), 971-976) describe the monitoring of growth curves of Escherichia coli and Staphylococcus epidermidis by Raman spectroscopy over extended periods of time (days) with the aim of identifying spectral variations throughout the growth cycle. In order to increase the signal to noise ratio, the fluorescence background signal, which is well-known to be present in biological samples, is bleached by prolonged laser irradiation, without affecting the Raman signal. In this document the Raman spectra of two strains belonging to different species and Gram-types are compared.
The technique of reducing the signal to noise ratio by bleaching the fluorescence background signal is also described by Esposito et al. (Appl. Spectrosc. 2003, 57(7), 868-871). Residual fluorescence can be removed by a polynomial fit. This is a mathematical procedure in which low frequency background signal, modelled by means of a polynomial function, fitted to the spectrum, is substracted from the Raman spectrum with fluorescent background. There is no guarantee that the shape of the polynomial resembles the actual shape of the fluorescence background. Although this document provides a number of spectra for individual spores that look very similar, it is noted that this document mentions that approximately 4% of the spores studied exhibited no Raman scattering attributable to calcium dipicolinate, and therefore yielded remarkably different Raman spectra. There is no suggestion in this document how to deal with this signal variance.
US-A-2005/0 185 178 describes the use of wide field Raman spectroscopy to quickly identify biological agents and pathogens. Photobleaching of substantial areas of the sample reduces the overall fluorescent background signal to enhance the Raman signal to noise ratio.
The vibrational spectroscopy-based microbial typing techniques described in the prior art, such as for instance disclosed in EP-A-1 623 212, comprise standardization of sample preparation. An important step is culturing of the patient material for a defined period of time on a standardized culture medium. After inoculation the micro-organisms in the patient sample will start to grow and divide and after a number of cell division cycles the cells will be in a growth condition that is independent of the condition of the micro-organism in the patient sample at the start of the culturing step. In this way, variance in the molecular composition of cells belonging to the same strain, but obtained from different sources, can be minimized.
However, standardization of culture conditions has been found to not always be sufficient to reduce intra-strain variance in molecular composition to the point where it no longer interferes with subspecies identification of micro-organisms by means of Raman spectroscopy. Differentiation of strains of microbial species based on their Raman spectra is then hindered because of the presence of molecular constituents of which the Raman signal contribution to the spectrum varies relatively strongly from one sample of a strain to the next sample of the same strain, despite the fact that the sample preparation procedure including the sample culturing have been standardized.
This is for instance the case for Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium strains. The Raman spectra of some of these strains show significant intra-strain qualitative variance in signal intensity, i.e. variations in relative signal intensity and shape of the bands of the spectra, e.g. throughout the 400 to 1800 cm−1 spectral region, hereafter referred to as the fingerprint region, between repeat cultures of the same strain.
Some of these signal variations are due to signal contributions of carotenoids, which can be present in abundance in S. aureus. Carotenoids generally show strong signal contributions at about 1004 cm−1, 1525 cm−1 and at 1157 cm−1. At these locations in the Raman spectrum, there is little overlap with signal contributions due to other molecular constituents, Therefore, if variance in signal intensity of these Raman bands affects the signal analysis, this can simply be remedied by eliminating these spectral regions from the analysis. However, it was found that spectra of S. aureus show significant signal variations essentially in all parts of the fingerprint region, from 400-1800 cm−1. This signifies the presence of other as yet unidentified constituents in S. aureus cells of which the concentration can vary significantly between repeated sample preparations. The resulting large variance in signal intensity of these molecular constituents negatively affects the reproducibility with which Raman spectra of S. aureus strains can be measured and thereby negatively affects the possibility to identify S. aureus isolates at the strain level.
With respect to hospital-acquired infections, methicillin resistant S. aureus (MRSA) strains are one of the largest problems in hospitals, worldwide. Any broadly applicable method for routine microbial typing must therefore be able to also type S. aureus strains.
Therefore, in order for vibrational spectroscopy to be applicable for routine strain-level identification of patient isolates, this problem must be resolved. Similar problems have been encountered in other species, e.g. Mycobacterium species. Therefore there exists an ongoing need for improving microbial typing techniques using vibrational spectroscopy.